Passive Immunotherapy of Viral Infections: 'Super-Antibodies'

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Passive immunotherapy of viral infections: ‘super-antibodies’ enter the fray

Laura M. Walker1 and Dennis R. Burton2–4 Abstract | Antibodies have been used for more than 100 years in the therapy of infectious diseases, but a new generation of highly potent and/or broadly cross-reactive human monoclonal antibodies (sometimes referred to as ‘super-antibodies’) offers new opportunities for intervention. The isolation of these antibodies, most of which are rarely induced in human infections, has primarily been achieved by large-scale screening for suitable donors and new single B cell approaches to human monoclonal antibody generation. Engineering the antibodies to improve half-life and effector functions has further augmented their in vivo activity in some cases. Super-antibodies offer promise for the prophylaxis and therapy of infections with a range of viruses, including those that are highly antigenically variable and those that are newly emerging or that have pandemic potential. The next few years will be decisive in the realization of the promise of super-antibodies.

The use of antibodies to ameliorate the adverse clini then the generation of fully human antibodies by vari cal effects of microbial infection can be traced back to ous techniques described below. Such antibodies have the late 19th century and the work of von Behring and been largely applied in the fields of oncology and auto Kitasato on the serum therapy of diphtheria and tetanus immunity. Only a single antiviral mAb, the RSV-specific (reviewed in REFS 1,2). In these settings, the antibodies antibody palivizumab, is in widespread clinical use. act to neutralize bacterial toxins. Therapies followed in The reasons for this have been discussed elsewhere1,3–6, which serum antibodies were targeted directly against although perhaps the most important reasons are the bacterial and then viral pathogens. For viral pathogens, fairly high cost of the production of mAbs, the difficul 1Adimab LLC, Lebanon, enriched polyclonal IgG molecules from immunized ties of administration and a belief that antibodies are New Hampshire 03766, USA. animals were shown to be effective in prophylaxis, largely effective only in a prophylactic setting, which can 2Department of Immunology and Microbiology, Center for and even prophylaxis after exposure, for a number of be achieved for many viruses by vaccination. HIV/AIDS Vaccine viruses, including hepatitis A virus, hepatitis B virus, However, as we discuss here, an increasing number Immunology and Immunogen hepatitis C virus, herpes simplex virus, measles virus, of antiviral antibodies with quite remarkable properties Discovery, The Scripps rabies virus, respiratory syncytial virus (RSV), smallpox in terms of potency and/or cross reactivity with other Research Institute, La Jolla, virus and varicella zoster virus. In general, the effective viruses or strains of the same virus are being isolated. California 92037, USA. 3International AIDS Vaccine ness of antibody preparations declined with the dura These so‑called super-antibodies are changing our Initiative Neutralizing tion of infection such that they were often regarded as understanding of what we can hope to achieve with anti Antibody Center, The Scripps poor therapeutic options. Of course, the major antiviral bodies against microbial infection in the clinic. Increased Research Institute, La Jolla, strategy of the 20th century was vaccination. potency can greatly reduce the unit costs of treatment, California 92037, USA. 4Ragon Institute of Over the latter part of the 20th and early part of make alternative routes of administration feasible and Massachusetts General the 21st century, there have been major developments extend the effective half-life of the antibody. Increased Hospital, Massachusetts in our understanding of antibodies and our ability to cross reactivity can allow us to consider targeting multi Institute of Technology and manipulate them. The advent of hybridoma technol ple viruses with single antibodies. Antibody engineering Harvard University, ogy in 1976 provided a reliable source of mouse mono can impact both potency and cross reactivity and can Cambridge, Massachusetts 02139, USA. clonal antibodies (mAbs), the first impact of which was greatly extend the half-life of super-antibodies. [email protected]; not on antibody therapy but on the characterization In this Review, we discuss how new approaches have [email protected] of cells through the definition of cell surface markers. fuelled the identification of super-antibodies, where doi:10.1038/nri.2017.148 Broad implementation of mAbs in therapy had to wait and how such antibodies may be best applied and future Published online 30 Jan 2018 until the development of humanized mouse antibodies and directions for the field.

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Super-antibody discovery Lassa viruses, extensive glycosylation on the surface Many acute viral infections induce robust neutralizing Env proteins results in masking of conserved neutral antibody responses in the large majority of individuals. izing epitopes15,32. In cases where super-antibodies are In general, these viruses show little evidence for evasion present at low frequency within immune repertoires, of antibody responses, and we have referred to them large-scale donor screening and high-throughput B cell as ‘evasion lite’ viruses7. Typically, they either display isolation platforms have proved to be critical for the dis limited antigenic variability in their surface protein (or covery of super-antibodies. Over the past several years, proteins) or show considerable variability but never technological advances in these two areas have led to theless express immunodominant, conserved epitopes. the identification of large numbers of super-antibodies, Examples of viruses in this category include measles mostly from infected individuals, against a plethora of virus, poliovirus, chikungunya virus and RSV8–11. The viral pathogens. life cycle of these viruses presumably does not dictate immune evasion. For these types of viruses, the iso Large-scale donor screening. In the case of HIV, which lation of super-antibodies from immune donors has has served as a prototype virus for many studies in this been achieved in a fairly straightforward manner8,9,12–14. field33, systematic selection of donors with broadly However, some viruses have evolved mechanisms to neutralizing serum responses has proved to be critical evade effective neutralizing antibody responses (termed for the identification of super-antibodies. Before 2009, ‘evasion strong’ viruses) and induce such responses at the HIV field had been operating with a handful of much lower levels. Effective responses in the context of bnAbs, all of which were limited either in breadth or in infection with highly antigenically variable viruses refers potency34. A number of factors complicated the iden not only to their neutralization potency but also to their tification of bnAbs, including the inefficiency of tradi effectiveness against diverse circulating global isolates, tional approaches to mAb discovery, the small fraction often referred to as breadth. For these viruses — includ of B cells that secrete bnAbs and the limited availability ing HIV, influenza virus, Ebola virus and Lassa virus of samples from donors who had developed broad and — only a proportion of infected individuals, sometimes potent neutralizing serum responses. Beginning in 2005, quite small, will generate broad and potent neutralizing the problem of limited samples was addressed by estab antibody responses15–21. Furthermore, within these indi lishing donor screening programmes to identify HIV- viduals, potent broadly neutralizing antibody (bnAb) infected individuals with broadly neutralizing serum specificities generally constitute only a small fraction of responses to serve as source material for the generation the antigen-specific memory B cell pool. of bnAbs18,19,35,36. In one of the largest studies, ~1,800 For example, only a small percentage of HIV‑infected HIV‑infected individuals from Australia, Rwanda, individuals develop broad and potent serum responses Uganda, the United Kingdom and Zambia were screened over time, and B cell cloning efforts have demonstrated for broadly neutralizing sera using a reduced pseudo that bnAbs generally comprise <1% of the HIV enve virus panel representative of global circulating HIV iso lope (Env)-specific memory B cell repertoire22. Although lates19. A subset of individuals, termed ‘elite neutralizers’, there are probably multiple factors that contribute to the was identified that exhibited exceptionally broad and low abundance of bnAbs within these individuals, the potent neutralizing serum responses and was therefore intrinsic nature of the viral Env protein likely has a key prioritized for bnAb isolation. role. The HIV Env protein has evolved a multitude of Over the past 8 years, mining of these and sim mechanisms to evade bnAb responses, including decoy ilar samples has led to the identification of dozens of forms of Env, enormous antigenic variability, an evolving remarkably broad and potent HIV super-antibodies37–41. glycan shield, immunodominant and variable epitopes Careful selection of donors with desirable serum profiles and poorly accessible conserved epitopes23. Furthermore, has also enabled the isolation of rare super-antibodies most HIV-specific bnAbs incorporate unusual features to influenza virus, RSV, human metapneumovirus for epitope recognition, such as uncommonly long (or (HMPV), rabies virus and Zika virus12,42–44. For exam short) complementarity determining region 3 (CDR3) ple, the pan-influenza A virus-neutralizing mAb FI6 and loops, insertions and deletions, tyrosine sulfation and the RSV and HMPV cross-neutralizing mAb MPE8 were extensive somatic hypermutation, that likely also con isolated from donors who were selected on the basis of tribute to the rarity and delayed development of bnAbs their strong heterotypic serum responses12,42. Similarly, during natural infection24–26. two pan-lyssavirus-neutralizing mAbs, called RVC20 In the case of influenza virus infection, the vast and RVC58, were isolated from the memory B cells of majority of neutralizing antibodies elicited by infec four donors who exhibited potent serum-neutralizing tion or vaccination bind to variable epitopes within the activity against multiple lyssavirus species43. haemagglutinin (HA) globular head of the viral parti 27 Humanized mouse cles and display strain-specific neutralizing activity . High-throughput human B cell isolation technologies. antibodies Influenza virus-specific bnAbs typically target the con Human antiviral neutralizing mAbs have been isolated Genetically engineered mouse served HA stem, but this region has variable immuno using various different technologies, including combina antibodies in which the protein genicity28–31. The relatively low frequency of bnAbs torial display libraries, human immunoglobulin trans sequence has been modified to (FIG. 1) increase its similarity to human against the HA stem is perhaps due to the typically tight genic mice and single B cell isolation methods . antibodies, thereby decreasing packing of HA trimers on the virus surface, which may Although all of these technologies have proved valuable its potential immunogenicity. limit antibody accessibility to this region. For Ebola and for mAb generation, the recent burst in super-antibody

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a Combinatorial display libraries b Humanized mice c Single B cell cloning

Human Human Human immunoglobulin Immunization donor transgenic mouse donor

Memory B cells Plasmablasts Human B cells

RT-PCR of V Antibody-forming cells Myeloma cells H + and VL genes (random pairing) Antigen Fusion Antigen-speciﬁc Plasmablast Phage Yeast Mammalian cell memory B cell sorting sorting Hybridoma cells CD38 Phage panning Limiting CD27 FACS selection dilution

1 2 3 4 5 6 7 8 9 10 11 12 A 1 2 3 4 5 6 7 8 9 10 11 12 B A C B D Hybridoma C E D Single- F screening E G F cell PCR H G H Clonal expansion Mammalian Mammalian cell cloning cell cloning and antibody Monoclonal and antibody expression antibodies expression

Figure 1 | Technologies for monoclonal antibody d Memory B cell immortalization e B cell culture generation. a | Combinatorial display libraries. Human antibody heavy-chain and light-chain genes are amplified by Human Human reverse transcription (RT)-PCR, and antibody fragments are donor donor displayed on the surface of a particle or cell in which the antibody genes are found (such as phage, yeast or mammalian cells142–145). Successive rounds of enrichment are performed to select for clones that bind to the target antigen. Genes encoding antibodies of interest are cloned into human Human Human IgG expression vectors to produce monoclonal antibodies B cells B cells (mAbs). b | Human immunoglobulin transgenic mice are generated by introducing human immunoglobulin loci into the mouse genome146,147. Upon immunization, the transgenic mice produce fully human antigen-specific antibodies. The Single-cell distribution

EBV 1 2 3 4 5 6 7 8 9 10 11 12 B cells harvested from the immunized mice are fused with A B myeloma cells to generate antibody-secreting hybridomas, C B cell activation D E and culture which are then screened for binding or functional activity. EBV CpG F G c | Single B cell cloning. Antigen-specific memory B cells or transformation H plasmablasts are single-cell sorted by flow cytometry, and 1 2 3 4 5 6 7 8 9 10 11 12 A cognate heavy-chain (Vh) and light-chain (Vl) variable genes B 57,58,148 C Binding or are amplified by single-cell PCR . The antibody variable D E functional F genes are cloned into human IgG expression vectors to G screening Limiting dilution H produce mAbs. d | Memory B cell immortalization. Memory

1 2 3 4 5 6 7 8 9 10 11 12 B cells are immortalized by Epstein–Barr virus (EBV), and A B Ampliﬁcation of VH and B cell culture supernatants are screened for binding or C D Screening VL genes from clones of interest 149 E functional activity . Positive cultures are subcloned by F G limiting dilution. e | Memory B cell culture. Single B cells are H activated and cultured, and B cell supernatants are screened for binding or functional activity37,150. Antibody variable genes Mammalian cell cloning are amplified from clones of interest by PCR and cloned into Monoclonal antibodies and antibody human IgG expression vectors to produce mAbs. FACS, expression fluorescence-activated cell sorting.

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discovery has primarily been driven by advances in isolated by testing 104,000 plasma cells from eight single B cell-based methods. There are several possi immune donors42. Finally, only 2 of 500 mAbs that were ble reasons for this, including inefficiencies in combi selected on the basis of their ability to neutralize rabies natorial library generation and interrogation (leading to virus showed cross-neutralizing activity against multiple the loss of rare clones), altered binding characteristics of lyssavirus species43. These examples clearly illustrate that antibody fragments produced in heterologous expres exhaustive interrogation of immune repertoires is often sion systems (for example, Escherichia coli or yeast), required for the identification of rare cross-neutralizing constraints on the generation of suitable recombinant super-antibodies. antigens for immunization or library selections, the loss A second breakthrough in the HIV antibody field of native heavy-chain and light-chain pairing during followed the development of technology for antigen- immune library generation and inherent differences specific single B cell sorting57–59. This approach, coupled between the adaptive immune systems of humanized with the use of rationally designed Env probes, allowed mice and humans45. for the discovery of two new potent HIV-specific bnAbs Several technological breakthroughs in the B cell that target the conserved CD4 binding site60,61 (TABLE 1). cloning arena have been most critical in fuelling Following this discovery, several other potent bnAbs super-antibody identification. One of these advances against the CD4 binding site were isolated through came in 2009, when direct functional screening of the use of similar approaches38,62,63. Recently, advances thousands of B cell clones from an HIV elite neutralizer in the generation of recombinant native-like HIV Env led to the isolation of two super-antibodies, PG9 and trimers have enabled the identification of exceptionally PG16, that were approximately an order of magnitude potent ‘PG9‑class’ bnAbs40. more potent than first-generation bnAbs37. Notably, Many HIV super-antibodies have now been gen PG9 and PG16 bind poorly to recombinant Env pro erated with the use of single B cell sorting technol teins and thus would not have been identified without ogy38,40,60,62,63. The use of fluorescently labelled probes direct functional screening of B cell supernatants. To to sort antigen-specific memory B cells has also ena date, many dozens of HIV-specific bnAbs targeting a bled the discovery of highly potent super-antibodies to diverse range of epitopes have been identified using Ebola virus, RSV, human papilloma virus (HPV), Zika functional screening approaches39,46–48. Highly potent virus and influenza virus11,30,31,44,64,65. In the case of Ebola super-antibodies to RSV, HMPV, Lassa virus and virus, a large-scale single B cell cloning effort led to the human cytomegalovirus (HCMV) have also been dis isolation of several hundred mAbs specific for Ebola covered using high-throughput functional screening virus envelope glycoprotein (GP), two of which showed technologies12,13,49,50 (TABLE 1). potent pan-Ebola virus-neutralizing activity and protec Similar to PG9 and PG16, these super-antibodies tive efficacy64,66. A similar effort in the RSV field allowed were isolated by screening B cell supernatants on the for the isolation of several prefusion F-protein-specific basis of their capacity to neutralize infection in vitro and mAbs that show over 100 times more potent neutral were subsequently found to react poorly with currently izing activity than palivizumab11. In addition, multiple available recombinant Env proteins. In the case of RSV, groups have used clever dual-antigen labelling strategies this approach led to the isolation of the highly potent to identify potent bnAbs to HIV, influenza virus, Ebola mAb D25, which binds to an epitope that is exclusively virus and HPV30,31,40,65,67,68. The structures of several expressed on the prefusion conformation of RSV fusion super-antibodies bound to their viral targets are shown glycoproteins (F proteins)49,51. An engineered variant of in FIGURE 2. Finally, a recent report showed that bnAbs mAb D25 (MEDI8897), which exhibits 50–100 times to HIV can be readily elicited in cows through the use greater neutralization potency than palivizumab, is of a single Env trimer immunogen and that this induc now being tested in clinical trials for the prevention of tion depends on the long heavy-chain CDR3 loops of the RSV-associated disease in high-risk infants. A second bovine immunoglobulin repertoire67. It is possible that RSV prefusion F protein‑specific super-antibody, which this repertoire may provide advantages in generating cross-neutralizes several different paramyxoviruses, super-antibodies against other pathogens. including HMPV, was also isolated by screening B cell Vaccination or infection-induced antibody-secreting supernatants for neutralizing activity12 (TABLE 1). cell (ASC) responses have also proved to be a rich source In the case of HCMV, direct functional screening of antigen-specific antibodies. Following early studies enabled the isolation of highly potent super-antibodies that showed that a transient but large population of specific for conformational epitopes within the gH–gL– ASCs appears in peripheral blood 5–7 days after tetanus UL128–UL130–UL131A pentamer complex, which was toxoid booster vaccination69, it was shown that influ not previously known to be a target for neutralizing anti enza virus vaccination produced a similar ASC response bodies13. Direct functional screening approaches have and that the large majority of mAbs cloned from these also led to the discovery of potent super-antibodies to cells bound with high affinity to influenza virus, provid Middle East respiratory syndrome coronavirus (MERS- ing a proof of concept that the ASC response could be CoV), Ebola virus, influenza virus, chikungunya virus, exploited to rapidly generate antigen-specific antibodies rabies virus and the poxvirus family9,42,43,52–56. Notably, against any immunizing antigen70. To date, plasmablast in the case of MERS-CoV, only 1 B cell culture out of cloning has led to the isolation of mAbs against many 4,600 screened showed neutralizing activity53. Similarly, different viruses, including dengue virus, Zika virus, the pan-influenza A virus-neutralizing mAb FI6 was HIV, influenza virus, vaccinia virus and rotavirus29,71–76.

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Table 1 | Antiviral super-antibodies Virus Prototypic super-antibody Similar antibodies* Antigenic region Antibody isolation technology HIV PG9 and PGT145 PG16, PGT141‑144, CH01‑04, V2 apex Human B cell isolation PGDM1400–1412 and CAP256‑VRC26.01‑12 VRC01 VRC02, VRC03, 8ANC131, 8ANC37, CD4 binding site Human B cell isolation 8ANC134, NIH45‑46, 3BNC60, BNC62, 3BNC117, 12A12, 12A21, 12A30, VRC‑PG04, VRC‑CH31, VRC27, VRC07‑523 and N6 PGT121, PGT128 and PGT135 PGT122, PGT123, PGT125‑PGT127, PGT130, V3 glycan Human B cell isolation PGT131, PGT136, PGT137, 10‑1074 and BG18 PGT151, 35O22 and 8ANC195 PGT152‑158, ACS202 and N123‑VRC34.01 gp120–gp41 interface Human B cell isolation 10E8 None identified MPER Human B cell isolation Influenza virus C05 F045‑092 and 641 I-9 HA head Human B cell isolation, phage display FI6 MEDI8852, CR9114, 39.29, 81.39, CT149, HA stem Human B cell isolation 56.a.09, 31.b.09, 16.a.26 and 31.a.83 RSV and MPE8 ADI‑14448 and 25P13 Site III Human B cell isolation HMPV RSV D25 AM22, 5C4 and ADI‑15618 Site ø Human B cell isolation HCMV 9I6 and 8I21 1F11, 2F4 and 6G4 Pentameric complex Human B cell isolation Rabies virus RVC58 None identified Site III Human B cell isolation RVC20 None identified Site I Human B cell isolation Dengue virus A11 and C8 C10, B2, B7 and C4 E‑dimer interface Human B cell isolation and Zika virus Z004 Z028, Z001, Z006, Z010, Z031, Z035, Z038, DIII lateral ridge Human B cell isolation Z014, ZKA‑190, ADI‑24192, ADI‑24232, ADI‑24227 and ADI‑24238 Ebola virus ADI‑15878 6D6, ADI‑15742, CA45 and FVM09 Fusion loop Human or macaque B cell isolation MERS-CoV LCA60, REGN3051 and None identified Receptor-binding Human B cell isolation, REGN3048 domain humanized mice Lassa virus 8.9F None identified Quaternary GPC‑C Human B cell isolation epitope 37.2D 25.6A Quaternary GPC‑B Human B cell isolation epitope 25.10C and 12.1F None identified Quaternary GPC‑A Human B cell isolation epitope *This list of antibodies is not exhaustive and is caveated by the fact that different neutralization assays can give different results. gp120, envelope glycoprotein gp120; gp41, glycoprotein 41; GPC, glycoprotein complex; HA, haemagglutinin; HCMV, human cytomegalovirus; HMPV, human metapneumovirus; MERS-CoV, Middle East respiratory syndrome coronavirus; MPER, membrane-proximal external region; RSV, respiratory syncytial virus.

One of the advantages of the plasmablast approach plasmablast response will be directed against immuno is that antigen baiting is not required for B cell sorting, dominant epitopes, which in many cases are not tar thereby allowing for the isolation of antibodies that geted by effective neutralizing antibodies. In such cases, target epitopes that are poorly presented on recom exhaustive cloning, production and characterization of binant antigens. For example, in the case of dengue the plasmablast-derived mAbs would likely be required virus, potent bnAbs targeting E‑dimer-dependent to identify rare super-antibodies. By contrast, secondary epitopes were isolated using this approach72. However, infection with an antigenically related but sufficiently it is important to emphasize that the ability to isolate divergent virus can drive the preferential expansion super-antibodies using this method will depend on of B cells that target highly conserved epitopes, as several factors. First, during a primary infection, the exemplified by the unusually high frequency of bnAbs ASC population will be mainly composed of activated, induced in donors who were infected with the novel low-affinity naive B cells (rather than affinity-matured H1N1 influenza virus in 2009 (REFS 29,75). In principle, memory B cells), making the possibility of identifying one could use this type of approach for the generation super-antibodies extremely unlikely. Second, in the of super-antibodies in humanized mice or other animal context of a booster vaccination or secondary infec models by using suitably designed immunogens and tion with an antigenically similar virus, most of the immunization regimens.

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a PGT145 b Inﬂuenza virus screening approach53. This mAb, called LCA60, showed HA protection both before and after exposure in a mouse model of MERS-CoV infection. Importantly, this process took the authors only 4 months from the initial B cell screening to the development of a stable cell line that produces the neutralizing mAb at 5 g l–1. In the second study, human immunoglobulin transgenic mice were immunized with the MERS-CoV spike protein and then HIV Env used to generate a panel of potent MERS-CoV-specific trimer neutralizing mAbs within several weeks77. The authors also quickly generated a humanized mouse model of CR9114 MERS-CoV infection, which was used to demonstrate the therapeutic efficacy of their mAbs. c RSV fusion d C8 In a third study, vaccination of transchromosomal trimer cows engineered to produce fully human IgG mole cules with MERS-CoV was shown to yield high serum titres of MERS-CoV-specific neutralizing antibodies78. Importantly, administration of the purified polyclonal transchromosomal bovine human IgG to mice either 12 hours before or 24 and 48 hours after MERS-CoV infection resulted in a significant reduction in viral lung titres. Transchromosomal bovines have also been used Zika virus envelope MPE8 to rapidly generate polyclonal neutralizing antibodies to Hanta virus, Venezuelan equine encephalitis virus and Nature Reviews | Immunology Figure 2 | Structures of super-antibodies bound to their target antigens. Ebola virus79–81, demonstrating the feasibility of using a | Cryoelectron microscopy structure of the broadly neutralizing anti-HIV‑1 this platform to rapidly generate therapeutics to com antibody PGT145 in complex with a recombinant HIV envelope (Env) trimer. PGT145 bat emerging viral threats. The antibodies arising from binds to a glycan-dependent quaternary epitope at the trimer apex 152. b | Crystal transchromosomal cows are polyclonal to date, but there structure of the influenza virus group 1 and group 2 neutralizing antibody CR9114 in complex with influenza virus haemagglutinin (HA). CR9114 recognizes a highly is potential for mAb isolation. conserved epitope in the HA stem154,155. c | Crystal structure of the respiratory Between 2015 and 2016, several groups responded syncytial virus (RSV) and human metapneumovirus cross-neutralizing antibody to the 2014–2015 Ebola virus outbreak by swiftly gener MPE8 in complex with a stabilized RSV prefusion fusion glycoprotein trimer. ating highly potent Ebola virus GP‑specific neutral d | Crystal structure of the Zika virus and dengue virus cross-neutralizing antibody izing mAbs from the memory B cells of convalescent C8 in complex with a soluble Zika virus Env ectodomain. C8 targets a quaternary donors54–56,64,66. Many of these mAbs showed potent epitope that bridges two Env protein subunits. Part a is adapted from REF. 151, therapeutic efficacy against either Ebola or Bundibugyo CC-BY-4.0. Part b is adapted with permission from REF. 153, AAAS. Part c is adapted virus after exposure in animal models and a subset from REF. 156, Macmillan Publishers Limited. Part d is adapted from REF. 85, showed protective efficacy against multiple Ebola virus Macmillan Publishers Limited. strains54,55,64,66. Similarly, several groups have recently reported on the isolation of potent Zika-virus-specific Rapid response platforms for emerging viruses neutralizing mAbs from human donors14,44,82,83. Notably, Over the past two decades, humanity has faced a newly one of these neutralizing mAbs, called ZIKV‑117, emerging, or re‑emerging, viral threat almost every year, showed protection against Zika virus after exposure in including severe acute respiratory syndrome coronavirus both pregnant and nonpregnant mice82. (SARS), West Nile virus, pandemic influenza virus, Ebola In certain cases, the availability of super-antibodies virus, MERS-CoV and Zika virus. Because of their com that target highly conserved epitopes may shorten time paratively fast path to approval and generally favourable lines further by bypassing the need for mAb discovery. safety profiles, mAb therapies represent a promising alter For example, it was recently shown that a subset of native to vaccines and small-molecule drugs for the treat dengue-virus-specific mAbs potently cross-neutralizes ment and prevention of emerging viral threats. Recently, Zika virus84–86. These bnAbs — perhaps carrying Fc several laboratories have demonstrated the feasibility of mutations that ablate Fc receptor binding to avoid the identifying, characterizing and scaling‑up production of potential for antibody-dependent enhancement14 — highly potent mAbs in remarkably short time frames. could immediately be used for prophylaxis for preg In response to the 2014–2015 MERS-CoV outbreak, nant women living in Zika-virus-endemic regions. two different groups illustrated the power of their mAb Notably, cocktails of super-antibodies targeting dif discovery platforms by isolating highly potent MERS- ferent epitopes, or bispecific or trispecific super-an Transchromosomal cows specific mAbs, producing the mAbs in gram quanti tibody constructs, will likely be required to prevent Cows that have been ties and testing the lead mAbs in animal models at an neutralization escape87–93. genetically modified to unprecedented speed53,77. In one of these studies, a sin incorporate human chromosomes so that upon gle highly potent MERS-CoV-neutralizing mAb was Antibodies in prophylaxis and therapy immunization they generate identified from the memory B cells of a convalescent Antibodies can function against viruses by several human antibodies. donor through the use of a high-throughput functional mechanisms, primarily divided into activities against

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free virus particles and activities against infected Although palivizumab is the only commercially cells. Neutralization, measured in vitro as the abil available mAb for the prevention of a viral disease, there ity of an antibody to prevent viral entry into target are multiple antiviral mAbs in preclinical and clinical cells without a requirement for involvement of any development that have shown efficacy prior to expo other agents, is an activity against free virions that sure in animal models. For example, the potent MERS- has been most correlated with protection in vivo. CoV-specific mAbs described above were shown to Activities against infected cells generally depend on prophylactically protect humanized mice against MERS- Fc effector functions and involve host effector cells. associated disease. Similarly, mAbs to chikungunya They include antibody-dependent cellular cytotoxicity virus, influenza virus, HIV and Ebola virus have shown (ADCC), complement-dependent cytotoxicity and potent prophylactic efficacy in animal models9,85,96–102. antibody-dependent cellular phagocytosis (ADCP). It Recently, a broadly neutralizing anti-Zika virus mAb is presumed that these activities are likely to be impor (ZIKV‑117) was shown to protect against maternal–fetal tant in antibody-based therapy. Because neutralization transmission in a mouse model of Zika virus infection82. frequently correlates with the ability to bind to native If this observation translates to humans, prophylaxis structures on the virion surface, it can give some indi with ZIKV‑117 or similar neutralizing mAbs may be a cation of the ability of antibodies to mediate effector promising means of protecting at‑risk pregnant women activities such as ADCC and ADCP. The potency of against Zika virus infection and fetal transmission. super-antibodies is then often estimated from neu In the case of HIV, multiple studies have shown tralization measurements, although ultimately it is of that passively administered neutralizing mAbs pro course in vivo activity that is crucial. vide protection against intravenous, vaginal, rectal Antibody-dependent and oral challenge in nonhuman primate and mouse cellular cytotoxicity Prophylaxis. Vaccination is the most effective and low- models99–101,103–106. A large ongoing study (the Antibody- (ADCC). A mechanism by which Fc receptor-bearing effector cost method of preventing viral disease. However, the Mediated Prevention (AMP) study will assess the abil cells such as natural killer (NK) development of effective vaccines against many impor ity of the VRC01 mAb specific for the CD4 binding cells recognize and kill tant viral pathogens — including HIV, RSV, hepatitis C site to decrease the risk of HIV acquisition in humans. antibody-coated target cells, virus, dengue virus and HCMV — has been met with Although animal studies have provided proof of prin such as virus-infected cells. The Fc portions of the coating limited success. Furthermore, vaccine development ciple that a vaccine capable of inducing sufficient titres antibodies interact with an Fc is a long complex process, often lasting 10–15 years, of bnAbs could prevent the establishment of HIV infec receptor (for example, FcγRIII; making immunization an impractical means of pro tion in humans, and the AMP study will investigate this which is expressed by NK tecting individuals from newly emerging viral threats directly, the design of immunogens that efficiently elicit cells), thereby initiating a unless pan-virus family vaccines can be developed. For these rare antibodies remains a formidable challenge. signalling cascade that results in the release of cytotoxic example, if antibodies can be identified that potently To bypass the challenges associated with active granules (containing perforin neutralize existing strains of Ebola virus, or even Ebola vaccination against HIV, a number of groups have and granzyme B) from the and Marburg filoviruses, then one could anticipate that proposed alternative strategies on the basis of vec effector cell, which lead to cell a vaccine templated from the antibodies would also tor-mediated antibody gene transfer to express bnAbs death of the antibody-coated 107 cell. be effective against emerging strains of Ebola virus. in vivo . Unlike traditional passive immunization, However, at the current time, passive antibody prophy which would require long-term repeated treatment Complement-dependent laxis represents a promising alternative to vaccination with bnAbs, vectored immunoprophylaxis involves cytotoxicity for a number of viral infections. only a single injection and enables continuous and A mechanism of Currently, three purified polyclonal hyperimmune sustained delivery of antibodies. In 2009, pioneering antibody-mediated immunity globulins whereby the association of an derived from human donors immune to hepa work demonstrated that vector-mediated delivery of antibody on a target cell titis B virus, HCMV or varicella zoster virus are on the antibody-like molecules can provide vaccine-like pro surface leads to binding of the market for the prevention of serious diseases associated tection against simian immunodeficiency virus (SIV) complement component C1q with these viruses. A rabies-virus-specific immune challenge in nonhuman primate models108. Subsequent and triggering of the classical complement cascade. The globulin, combined with vaccination, is also available studies have shown that vectored immunoprophylaxis cascade leads to elimination of for prophylaxis after exposure. In 1998, palivizumab is also compatible with full-length IgG molecules and target cells by a number of — a humanized mAb that targets the RSV F protein — CD4‑like molecules109–111. If the preclinical results in mechanisms, including the became the first antiviral mAb approved by the US Food mice and macaques translate to humans, vectored formation of the membrane and Drug Administration. Palivizumab soon replaced antibody gene delivery strategies could provide an attack complex, the cytolytic end product of the the RSV hyperimmune globulin (RespiGam) for the alternative form of prophylaxis against HIV and other complement cascade. prevention of severe RSV-associated disease in high- challenging vaccine targets, such as hepatitis C virus, risk infants. However, although palivizumab is more pandemic influenza virus and malaria. Recently, non Hyperimmune globulins specific and 50–100 times more potent than RespiGam, viral vector nucleic acid delivery technologies have also Antibody preparations generated from plasma of the cost associated with the required dosing makes its been developed to obviate the potential safety issues 94 donors with high titres of an use impractical for all infants . A second-generation associated with viral vector-mediated delivery, such as antibody against a specific RSV-specific mAb, which shows up to 50 times greater long-term persistence and potential viral DNA integra pathogen or antigen. neutralization potency than palivizumab and contains tion into the host genome112–117. In a recent study, it was Hyperimmune globulins are substitutions in the Fc domain that extend its serum shown that the administration of lipid-encapsulated available against rabies virus, hepatitis B virus and varicella half-life, is currently in phase II clinical trials for the pre nucleoside-modified mRNAs encoding the heavy- 95 zoster virus, among other vention of severe RSV-associated disease in all infants chain and light-chain genes of the broadly neutral viruses. (TABLE 2). izing HIV‑1‑specific antibody VRC01 to humanized

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Table 2 | Antiviral monoclonal antibodies in clinical development Antibody Virus Antibody isolation Target Stage of Manufacturer Indication technology development Porgaviximab Ebola virus Immunization and Viral Env Phase I and II Mapp Bio Treatment of Ebola virus chimerization glycoprotein pharmaceutical; LeafBio infection after exposure MBL HCV1 HCV Humanized mice HCV E2 Phase II MassBiologics Prevention of HCV glycoprotein recurrence in patients receiving a liver transplant PRO 140 HIV Immunization and CCR5 Phase III Progenics Treatment of HIV‑1 infection humanization Pharmaceuticals Ibalizumab HIV Immunization and CD4 Phase III TaiMed Biologics Treatment of HIV‑1 infection humanization UB 421 HIV Immunization and CD4 Phase II United Biomedical Treatment of HIV‑1 infection humanization VRC01‑LS HIV Human B cell isolation HIV gp120 Phase I National Institute of Prevention of HIV‑1 Allergy and Infectious infection Diseases VRC01 HIV Human B cell isolation HIV gp120 Phase I National Institute of Treatment of HIV‑1 infection Allergy and Infectious Diseases 3BNC117‑LS HIV Human B cell isolation HIV gp120 Phase I Rockefeller University Treatment of HIV‑1 infection 10‑1074 and HIV Human B cell isolation HIV gp120 Phase I Rockefeller University Treatment of HIV‑1 infection 3BNC117 PGT121 HIV Human B cell isolation HIV gp120 Phase I International AIDS Treatment and prevention of Vaccine Initiative HIV‑1 infection PGDM1400 HIV Human B cell isolation HIV gp120 Phase I International AIDS Treatment and prevention of and PGT121 Vaccine Initiative HIV‑1 infection MB 66 HIV and Human B cell isolation HIV gp120 Phase I Mapp Prevention of HIV‑1 and HSV HSV and HSV Biopharmaceutical sexual transmission glycoprotein D VIS 410 Influenza Unknown Influenza virus HA Phase II Visterra Treatment and prevention of virus influenza A virus infection MHAA Influenza Human B cell isolation Influenza virus HA Phase II Genentech Treatment of influenza A 4549A virus virus infection CT P27 Influenza Human B cell isolation Influenza virus HA Phase II Celltrion Treatment and prevention of virus influenza A virus infection Diridavumab Influenza Phage display Influenza virus HA Phase II National Institute of Treatment and prevention of virus Allergy and Infectious influenza A virus infection Diseases CR8020 Influenza Human B cell isolation Influenza virus HA Phase II Crucell Treatment and prevention of virus influenza A virus infection RG 6024 Influenza Human B cell isolation Influenza virus HA Phase I Genentech Treatment of influenza B virus virus infection MEDI 8852 Influenza Human B cell isolation Influenza virus HA Phase II MedImmune Treatment of influenza A virus virus infection TCN 032 Influenza Human B cell isolation Influenza virus Phase II Theraclone Sciences; Treatment of influenza A virus M2e protein Zenyaku Kogyo virus infection m 102.4 Nipah and Phage display Viral Env Phase I Profectus Biosciences, Prevention and treatment Hendra glycoprotein G Inc. of Nipah and Hendra virus virus infections Rabimabs Rabies virus Immunization Viral Env G protein Phase I and II World Health Treatment and prevention Organization; Zydus of rabies Cadila RAB‑1 Rabies virus Humanized mice Viral Env G protein Approved Serum Institute of Prophylaxis after exposure India; MassBiologics to rabies Foravirumab Rabies virus Phage display, human Viral Env G protein Phase II and Crucell; Sanofi Pasteur Prophylaxis after exposure B cell isolation III to rabies Palivizumab RSV Immunization and Viral fusion Approved MedImmune Prophylaxis in high-risk humanization protein infants MEDI 8897 RSV Human B cell isolation Viral fusion protein Phase II MedImmune Prophylaxis in all infants CCR5, CC-chemokine receptor 5; E2, envelope glycoprotein (HCV); Env, envelope; gp120, envelope glycoprotein gp120; HA, haemagglutinin; HCV, hepatitis C virus; HSV, herpes simplex virus; M2e, matrix protein 2 (influenza virus); RSV, respiratory syncytial virus.

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mice resulted in high serum antibody concentrations targets an epitope overlapping that bound by palivi and protection against intravenous HIV‑1 challenge114. zumab recently showed a trend towards a therapeutic Similar proof‑of‑concept studies have also been per effect in a phase I and IIa clinical trial based on reduced formed using synthetic DNA plasmid-mediated anti viral loads and clinical symptoms in hospitalized RSV- body gene transfer112,113. In one such study, synthetic infected infants. Prefusion F-protein-specific sdAbs DNA plasmids encoding cross-neutralizing anti-dengue that show up to 180,000 times greater neutralization virus antibodies were delivered to mice by electro potency than ALX‑0171 have recently been identified poration and resulted in biologically relevant levels of and may offer even greater therapeutic benefit133. serum antibody112. Importantly, a single intramuscular injection of plasmid DNA conferred protection against Practical considerations severe dengue disease in a mouse model. Although Intuitively, the enhanced potency of super-antibodies several technical challenges remain to be addressed, is immediately recognized as beneficial in antibody such as enhancing in vivo antibody expression levels prophylaxis and therapy. However, there are also and reducing the potential for immunogenicity, these a number of additional effects from this enhanced studies demonstrate the feasibility of utilizing plasmid potency that may not be instantly appreciated and that DNA and modified mRNA-based antibody delivery can be further strengthened by antibody engineer technologies for passive immunotherapy. ing. For example, enhanced potency means that less antibody needs to be used, and this can enable easier- Therapy. Conventional wisdom says that antibodies to-develop, low-concentration subcutaneous adminis are effective if present before or shortly after viral tration rather than the use of more-difficult-to-develop, exposure, but their effectiveness declines markedly high-concentration subcutaneously administered once infection is established. For example, the anti- formulations or less-convenient (low-concentration) RSV antibody palivizumab is effective in the clinic intravenous administration. Enhanced potency also prophylactically but not therapeutically118. However, means that the lifetime of an effective antibody follow there are indications that the dogma may be chal ing administration is extended, thereby requiring fewer lenged by super-antibodies. An example is the ability administrations to maintain a useful protective or thera of a new generation of bnAbs against HIV to strongly peutic effect. Antibody engineering can also extend impact ongoing infection in animal models87,119,120 its half-life considerably95,134–139 so that for the most in which an earlier generation of less-potent mAbs potent super-antibodies, one could envisage requiring had very limited effects121. This ability likely reflects administrations perhaps only every 3–6 months for the increased neutralization potency of the super- effectiveness. Antibody engineering can also deliver antibodies as well as the increased breadth of neutral greater effectiveness through enhanced Fc effector ization that may restrict virus escape pathways119. A function140,141. number of super-antibodies are now being evaluated in humans for their activities against established HIV Conclusions infection122–126 (TABLE 2). Initial results are interesting, The deployment of antibodies as antiviral agents providing for example an indication of enhanced has progressed through a number of stages over the immune responses following bnAb administration127. years, corresponding to increasing levels of potency The emerging results will be followed closely, includ of the reagent administered. Passive immunotherapy ing in the context of combining bnAbs with drugs and began with immune serum over a century ago, then other antiviral agents to attempt HIV cure. progressed to polyclonal antibodies, then mAbs and For other viruses, clear evidence of a strong thera now into highly potent human mAbs dubbed super- peutic effect for super-antibodies has not been gath antibodies. Thanks to research that has been primarily ered yet. Several cases such as antibody treatment of carried out in the field of cancer research, technologies rabies virus and Junin virus infections43,128 are probably have been developed to endow these super-antibodies better interpreted as prophylaxis after exposure rather with enhanced in vivo function. than therapy for an established infection. Two prom Will super-antibodies change the landscape of ising examples of possible therapy are the successful antiviral prophylaxis and therapy? The answer to this treatment of Ebola-virus-infected or Lassa-virus- question will depend on a number of factors: first, the infected monkeys with mAbs once symptoms have rapidity of development of antiviral vaccines (vaccines appeared89,129. Unfortunately, no definitive evidence will likely remain the least expensive and most effec of the effectiveness of mAbs in Ebola-symptomatic or tive antiviral measure, but some viruses such as HIV Lassa-symptomatic humans yet exists. present a large challenge to vaccine development); sec Camelid-derived single-domain antibodies (sdAbs), ond, the effective cost of antibody treatment, which which contain a single heavy-chain variable domain, incorporates not only manufacturing cost but also represent a promising new class of antibody-based the durability of the administered antibody and the therapeutics for RSV and other viruses that cause lower route of administration; and third, the success of anti respiratory tract infections130–133. Because of their small bodies in the treatment of established viral infections. size and high solubility and stability, sdAbs can be rap Particularly in the therapeutic setting, the answers idly delivered to the site of infection via inhalation. can only be obtained with clinical trials using the best Notably, a neutralizing anti-RSV sdAb (ALX‑0171) that super-antibodies available.

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